Pituitary Adenylate Cyclase-Activating Peptide (PACAP)-Glutamate Co-transmission Drives Circadian Phase-Advancing Responses to Intrinsically Photosensitive Retinal Ganglion Cell Projections by Suprachiasmatic Nucleus

Peder T Lindberg,Sunny Z Jiang,Martin K.-H Schäfer,Lee E Eiden,Eberhard Weihe,Martha U Gillette,Christian Beaulé,Jennifer W Mitchell,Penny W Burgoon

doi:10.3389/fnins.2019.01281

Abstract

Results from a variety of sources indicate a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in light/glutamate-induced phase resetting of the circadian clock mediated by the retinohypothalamic tract (RHT). Attempts to block or remove PACAP’s contribution to clock-resetting have generated phenotypes that differ in their responses to light or glutamate. For example, previous studies of circadian behaviors found that period-maintenance and early-night phase delays are intact in PACAP-null mice, yet there is a consistent deficit in behavioral phase-resetting to light stimulation in the late night. Here we report rodent stimulus–response characteristics of PACAP release from the RHT, and map these to responses of the suprachiasmatic nucleus (SCN) in intact and PACAP-deficient mouse hypothalamus with regard to phase-resetting. SCN of PACAP-null mice exhibit normal circadian rhythms in neuronal activity, but are “blind” to glutamate stimulating phase-advance responses in late night, although not in early night, consistent with previously reported selective lack of late-night light behavioral responsiveness of these mice. Induction of CREB phosphorylation, a hallmark of the light/glutamate response of the SCN, also is absent in SCN-containing ex vivo slices from PACAP-deficient mouse hypothalamus. PACAP replacement to the SCN of PACAP-null mice restored wild-type phase-shifting of firing-rate patterns in response to glutamate applied to the SCN in late night. Likewise, ex vivo SCN of wild-type mice post-orbital enucleation are unresponsive to glutamate unless PACAP also is restored. Furthermore, we demonstrate that the period of efficacy of PACAP at SCN nerve terminals corresponds to waxing of PACAP mRNA expression in ipRGCs during the night, and waning during the day. These results validate the use of PACAP-deficient mice in defining the role and specificity of PACAP as a co-transmitter with glutamate in ipRGC-RHT projections to SCN in phase advancing the SCN circadian rhythm in late night.

Highlights

Internal circadian clocks must be both self-sustaining and flexible if they are to be useful in organizing behavior and physiology
Applying glutamate to suprachiasmatic nucleus (SCN) of wild-type and pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice is an approximation of natural phase shifting, but one step further “downstream” from simple light exposure
To test whether or not PACAP-null mice would show intact shifts to direct glutamate application, we examined the glutamate responsiveness of wild-type and PACAP null mice at selected time-points of the early (CT 14) and late (CT 22) night, to determine if the PACAP dependence of light-induced phase advance in circadian rhythm we reported in intact mice would persist in the ex vivo preparation

Summary

Introduction

Internal circadian clocks must be both self-sustaining and flexible if they are to be useful in organizing behavior and physiology. Despite discoveries regarding peripheral oscillators, the circadian clock in the SCN is still considered one of the few sets of mammalian cells that maintains its own circadian rhythm relative to the external environment. It does this by responding to external time cues, the most important of which for mammals is light. This combination of internal rhythm generation and sensitivity to change makes the SCN an intriguing model of intrinsic mammalian brain function, and its adaptation to the environment. PACAP has been shown to be associated with melanopsin, one of photopigments of the retina necessary for clock entrainment (Panda et al, 2002; Ruby et al, 2002)

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